Solid Support Having Ligand Immobilized Thereon By Using Photocleavable Linker
a technology of immobilization and solid support, which is applied in the field of solid support having a ligand immobilized thereon, and can solve the problems of difficult elution and recovery of selective binding proteins alon
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example 1
Synthesis of Resin on which FK506 is Immobilized via a Photocleavable Linker
[0067] A mixture of a photocleavable linker (4-{4-[1-(Fmoc-amino)-ethyl]-2-methoxy-5-nitrophenoxy}butyric acid, Fluka) (124 mg, 0.24 mmol), TOYOPEARL resin (TSKgel AF-amino, 600 μl, free amino group (available amino group) was 0.06 mmol), EDC (44 mg, 0.28 mmol), HOBt (39 mg, 0.28 mmol) and DMF (6 ml) was stirred at room temperature for 15 hr. The reaction end point was confirmed by the incapability of visual observation of residual amino group in the ninhydrin reaction. The reaction rate at that time was calculated to be about 88%. After confirmation of the completion of reaction, the resin was washed 5 times with DMF. Thereto was added 20% piperidine-DMF solution (6 ml) and the mixture was stirred at room temperature for 2 hr. The resin was washed 5 times with DMF, and 200 μl thereof was taken and stirred with a mixture of 17-allyl-1, 14-dihydroxy-12-{2-[4-(7-carboxy-heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-...
example 2
(1) Preparation of Rat Brain Lysate
[0068] The rat brain (2.2 g) was mixed in a mixture A (0.25M sucrose, 25 mM Tris buffer (pH 7.4), 22 ml) and prepared as a homogenate, which was then centrifuged at 9500 rpm for 10 minutes. The centrifugal supernatant was collected and further centrifuged at 50000 rpm for 30 minutes. The supernatant thus obtained was used as the lysate. Note that the experiment was entirely conducted at 4° C. or on ice.
(2) Binding Assay and Purification of Target Protein
[0069] A resin (TOYOPEARL AF) on which FK506 was immobilized via a photocleavable linker (4-{4-[1-(Fmoc-amino)-ethyl]-2-methoxy-5-nitrophenoxy}butyric acid, Fluka), which was prepared in Example 1, was mixed with a rat brain lysate prepared in the above-mentioned (1), and the attached product was washed with buffer B (25 mM tris buffer (pH 7.4), 0.25 M sucrose, 500 mM hydrazine, 500 mM 2-ME). For elution thereafter, the resin was subjected to photoirradiation in buffer B for 1 hr using a large ...
example 3
Synthesis of Resin on which Cromoglycic acid is Immobilized via a Photocleavable Linker
[0072] A mixture of a resin (500 μl), on which a photocleavable linker was bound, which was prepared by the method described in Example 1, cromoglycic acid (93 mg, 0.2 mmol), EDC (37 mg, 0.24 mmol), HOBt (32 mg, 0.24 mmol) and NMP (5 ml) was stirred at room temperature for 20 hr. The reaction end point was confirmed by the incapability of visual observation of residual amino group in the ninhydrin reaction. The reaction rate at that time was calculated to be about 76%. After confirmation of completion of the reaction, the resin was washed 5 times with NMP. Acetic anhydride (1 ml) and NMP (4 ml) were added thereto, and the mixture was stirred at room temperature for 1 hour. Subsequently, the resin was thoroughly washed with NMP, and the obtained resin immobilizing cromoglycic acid via photocleavable linker was used for the binding assay described below.
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